Biosafety and Proteome Profiles of Different Heat Inactivation Methods for Mycobacterium tuberculosis

Abstract

Studies involving the pathogenic organism Mycobacterium tuberculosis routinely require advanced biosafety laboratory facilities, which might not be readily available in rural areas where tuberculosis burdens are high. Attempts to adapt heat inactivation techniques have led to inconsistent conclusions, and the risk of protein denaturation due to extensive heating is impractical for subsequent mass spectrometry (MS)-based protein analyses. In this study, 240 specimens with one or two loops of M. tuberculosis strain H37Rv biomass and specific inactivated solutions were proportionally assigned to six heat inactivation methods in a thermal block at 80°C and 95°C for 20, 30, and 90 min. Twenty untreated specimens served as a positive control, and bacterial growth was followed up for 12 weeks. Our results showed that 90 min of heat inactivation was necessary for samples with two loops of biomass. Further protein extraction and a matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) MS assay demonstrated adequate scores for bacterial identification (≥1.7), with the highest score achieved in the 80°C/90 min and 95°C/30 min treatment groups. A proteomics study also confidently identified 648 proteins with ∼93% to 96% consistent protein abundances following heating at 95°C for 20, 30, and 90 min. Heat inactivation at 95°C for 90 min yielded the most quantifiable proteins, and a functional analysis revealed proteins located in the ribosomal subunit. In summary, we proposed a heat inactivation method for the M. tuberculosis strain H37Rv and studied the preservation of protein components for subsequent bacterial identification and protein-related assays. IMPORTANCE Inactivation of Mycobacterium tuberculosis is an important step to guarantee biosafety for subsequent M. tuberculosis identification and related research, notably in areas of endemicity with minimal resources. However, certain biomolecules might be denatured or hydrolyzed because of the harsh inactivation process, and a standardized protocol is yet to be determined. We evaluated distinct heating conditions to report the inactivation efficiency and performed downstream mass spectrometry-based M. tuberculosis identification and proteomics study. The results are important and useful for both basic and clinical M. tuberculosis studies.

Keywords: Mycobacterium tuberculosis; biosafety; heat inactivation; mass spectrometry; proteomics.

FIG 1

(Ai and Aii) Growth performances of the first set of samples subjected to the six inactivation processes. Growth evaluations were performed in both MGIT broth culture and LJ medium culture. (Bi and Bii) Growth performances of the second set of samples subjected to the six inactivation processes. Data are presented as the percent (%) of total colonies versus the day of observation. The total observation period is 84 days (12 weeks).

FIG 2

MALDI-TOF mass spectra of Mycobacterium tuberculosis (MTB) heat-inactivated samples treated with different temperatures and durations.

FIG 3

Principal-component analysis of MALDI-TOF results. (Ai to Aiii) Comparison of different heat inactivation temperatures. (Bi and Bii) Comparison of different heat inactivation times. (C) Comparison of the six heat inactivation conditions.

FIG 4

(A) Overlap of protein identification of three different heat inactivation methods and comparison of the common protein regulation. Upregulated and downregulated proteins were defined as ≥2 and ≤2 multiples of change, respectively. Annotation of (B) biological processes, (C) molecular functions, and (D) cellular components of the commonly quantified proteins (blue bars) and proteins uniquely quantified in the 90-min group (gray bars) by Gene Ontology analyses.